Characterizing the sequence diversity of

ACC deaminase of rhizobacterial strains

associate with leguminous plants of arid,

semi arid regions of Rajasthan

Presented by:-

Suman Kumar

Deptt:-Biotechnology

2013MSBT022

Legumes are members of the plant family Fabaceae

Legume plant are important as they constitute one of the

richest source of dietary source of proteins.

Legume crops constitute one of the most important

commercial crops of arid, semi- arid regions of Rajasthan.

Rhizosphere- Soil layer bound to the plant root. It is a

zone of many beneficial plant - microbe interactions.

Rhizobacteria- Soil bacteria residing in the rhizosphere

are termed as rhizobacteria.

Some rhizobacteria are capable of enhancing plant growth

and they are called

Plant growth promoting rhizobacteria (PGPR)

Modified from: XiteBio Technology Inc.

Plant stress control

Salinity stress

pH stress

Heavy metals stress

Organic contamination stress

Extreme temperature stress

ACC (1-Aminocyclopropane-1-carboxylase) deaminase

10/13/2015

• Whether this mechanism also exist in Legume plants

growing in arid, semi- arid regions of Rajasthan?

• If so, what is the sequence diversity of ACC deaminase

present in PGPRs associated with legume plants of arid,

semi- arid regions of Rajasthan?

Diversity of rhizobacterial strains isolated from

leguminous plants of arid, semi arid regions of Rajasthan.

Soil sample collection and processing

Determining the of physico-chemical characteristics

of soil samples

Isolation, purification and

preservation of culturable bacterial

isolates from legume rhizosphere

Optimization of soil

DNA procedure

Soil DNA PCR

RFLP analysis of acdS amplicons

Optimization of colony PCR for

acdS amplification

Sequencing of representative acdS amplicons

Sample

AreaSample Name

(Legume

Plants)

No. of

collected

Sample

Temperature

during Sampling

Crop age at time

of sample

collection

Village- Untwaliya

District- Churu

State-Rajasthan

(Altitude: 75.02)

(Latitute: 28.29oN)

(Longitude: 74.96oE)

Tilhan 10 Env-38ºC

Soil-37ºC

90 Days

Gaur 10 Env-40ºC

Soil-38ºC

90 Days

Mung 10 Env-43ºC

Soil-41ºC

90 Days

Moth 5 Env-42ºC

Soil-42ºC

90 Days

Farmer Name:- Mula Ram &

Sharvan Kumar.

Sample

Name

(Legume

Plants)

Soil type

No. of

irrigation

times

Fertilizer

used

Pesticide

used

Harvesting Time

Tilhan Arid and

Semi-arid

2 Rain No No Late OCT-2014

Gaur Arid and

Semi-arid

3Rain Yes,

Only

Compost

No Mid NOV-2014

Mung Arid and

Semi-arid

1 Rain Yes,

Only

Compost

No Late OCT-2014

Moth Arid and

Semi-arid

1Rain Yes,

Only

Compost

No Late OCT-2014

Sample collection report Farmer Name:- Mula Ram &

Sharvan Kumar.

pH analysis of soil samples Organic carbon result of the soil samples

Physico-chemical

characteristic

Samples Name

Guar

Rhizosphere

soil

Moong

Rhizosphere

soil

Moth

Rhizosphere

soil

Tilhan

Rhizosphere

soil

pH ~ 9 ~ 9 ~ 9.5 ~ 9

Oxidizable

Organic

carbon

Medium-Low

(0.300-0.500)

Medium-Low

(0.300-0.500)

High

(1.001-50)

Medium-Low

(0.300-0.500)

Phosphate 22-56kg/hac 22-56kg/hac 22-56kg/hac 22-56kg/hac

Potassium Medium

112-280kg/hg

High

280-390kg/hg

Low

112kg/hg

High

280-390kg/hg

Ammonical

Nitrogen

Medium

About-15kg/hg

Medium

About-15kg/hg

Medium

About-15kg/hg

Medium

About-15kg/hg

Nitrate

nitrogen

Very Low

About-4kg/hg

Very Low

About-4kg/hg

Very Low

About-4kg/hg

Very Low

About-4kg/hg

1. Spreading of diluted samples

on Nutrient Agar medium

1. 2.Purification of bacterial isolates

on Nutrient Agar medium

All Soil Samples were isolate and purified on Nutrient agar media.

Total 232 rhizobacterial isolate are purified

M.G.R.001,(44) M.G.S.001,(76) M.R.001,(40) M.S.001,(34) T.R.001,(36)

T.S.001,(40) G.R.(36) G.S.001,(70) M.S.002,(32) T.S.002,(56).

M.S 001sample result

MG.R 001 sample result

s. no SIZE SHAPE COLOR TEXTURE HIGHT MARGIN

1 5 MM & MEDIUM SIZE IRREGULAR CREMISH & OPAQUE MUCOID & STICKY RISED ROUGH MARGIN

2 3-4 MM & SMALL SIZE IRREGULAR YELLOWISH & OPAQUE MUCOID & STICKY RISED ROUGH MARGIN

3 4-5 MM & MEDIUM SIZE IRREGULAR YELLOWISH & OPAQUE MUCOID RISED ROUGH MARGIN

4 4-5 MM & MEDIUM SIZE NON-CIRCULAR YELLOWISH & OPAQUE MUCOID & STICKY FLAT SMOTH MARGIN

5 6-7MM & MEDIUM LARGE IRREGULAR CREMISH & OPAQUE MUCOID FLAT FIBROUS MRGIN

6 5 MM & MEDIUM SIZE NON-CIRCULAR DULL YELLOWISH & OPAQUE MUCOID & STICKY FLAT SMOTH MARGIN

7 4-5 MM & MEDIUM SIZE NON-CIRCULAR CREMISH & OPAQUE MUCOID & STICKY FLAT SMOTH MARGIN

8 5-7 MM & MEDIUM LARGE NON-CIRCULAR DULL YELLOWISH & OPAQUE MUCOID & STICKY FLAT SMOTH MARGIN

9 3-4 MM & SMALL SIZE NON-CIRCULAR DARK YELLOWISH & OPAQUE MUCOID & STICKY FLAT SMOTH MARGIN

10 4-5 MM & MEDIUM SIZE NON-CIRCULAR DULL YELLOWISH & OPAQUE MUCOID & STICKY RISED ROUGH MARGIN

11 6-7 MM & MEDIUM LARGE NON-CIRCULAR DARK YELLOWISH & OPAQUE MUCOID & STICKY RISED ROUGH MARGIN

12 1-2 MM & SMALL SIZE NON-CIRCULAR YELLOWISH & OPAQUE MUCOID & STICKY RISED ROUGH MARGIN

13 4-5 MM & MEDIUM SIZE NON-CIRCULAR YELLOWISH & OPAQUE MUCOID FLAT SMOTH MARGIN

14 4-5 MM & MEDIUM SIZE NON-CIRCULAR ORANGESIH & TRANSLUCENT MUCOID RISED SMOTH MARGIN

15 5MM & MEDIUM SIZE NON-CIRCULAR YELLOWISH & OPAQUE MUCOID RISED SMOTH MARGIN

s.no SIZE SHAPE COLOR TEXTURE HIGHT MARGIN

1 8-9 MM & LARGE SIZE RHIZOID CREMISH & TRANSLUCENT MUCOID FLAT FIBROUS

2 7-8 MM & LARGE SIZE CIRCULAR CREMISH & OPAQUE MUCOID FLAT SMOOTH MARGIN

3 1-1.5 MM & VERRY SMALL CIRCULAR CREMISH TRANSLUCENT MUCOID RISED SMOOTH MARGIN

4 5-6 MM&LARGE SIZE CIRCULAR CREMISH & OPAQUE MUCOID DRIED FLAT SMOOTH MARGIN

5 6-8MM & LARGE SIZE CIRCULAR DULL CREMISH & OPAQUE MUCOID STICKY FLAT SMOOTH MARGIN

6 6-7MM & LARGE SIZE NON-CIRCULAR DULL CREMISH & OPAQUE MUCOIDSTICKY FLAT ROUGH MARGIN

7 3-4 MM & MEDIUM SIZE IRREGULAR CREMISH & OPAQUE MUCOID STICKY FLAT FIBROUS MARGIN

8 3-4 MM & MEDIUM SIZE IRREGULAR CREMISH & OPAQUE MUCOID STICKY FLAT ROUGH MARGIN

1 2 3 4

Lane 1- DNA ladder 1kb

Lane 2- blank

Lane 3- Moong soil samples

S.NO. Name of sample DNA µg/ml

1 Moong sample 0.13 µg/ml

2 Gaur sample 0.11 µg/ml

5 6

Lane 4- gaur soil sample

Agarose gel analysis of soil DNA

Nanodrop spectrophotometer data

Lane 1- DNA ladder 1kb

Lane 2- blank

Lane 3- Garden soil samples

S.NO. Name of sample DNA µg/ml

1 Garden soil sample 31µg/ml

2 Gaur soil sample 18.01µg/ml

3 Moong soil sample 15µg/ml

Lane 4-5- Gaur soil sample

Lane 6-7- Moong soil sample

Agrose gel analysis in soil DNA isolation

1 2 3 4 5 6 7

Lane 3-7: Soil DNA PCR products for acdS

Agrose gel analysis of PCR

products from PCR for acdS

1 2 3 4 5 6 7

Lane 1: DNA ladder

Lane 2: blank

Based on the results obtained, we conclude that amicrobial collection of ~ 235 culturable bacterial isolatesassociated with rhizosphere of legume crops of arid andsemi- arid region of Rajasthan have been isolated.

These culturable isolates as well as rhizospheric soil DNAwere tested with PCR for presence of acdS.

However, due to lack of optimized protocols, it could notget accomplished.

The isolates will be screened for their role in other

plant growth promoting activities.

Diversity of ACC deaminase gene these isolates and

unculturable diversity would be characterized with

PCR and cloning base approaches.

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